Project Summary Our goal is to validate a novel Lactococcus lactis (L. lactis)-based platform to rapidly develop and deliver tolerance-inducing therapeutics for the treatment of autoimmunity and allergy. Autoimmune and allergic diseases, such as Multiple Sclerosis (MS), Type 1 Diabetes, and food allergies affect 25-50% of the world population, and are continuing to rise1-3. These illnesses are the result of the loss of tolerance to self or environmental antigens and cause symptoms ranging from minor irritation to debilitation or death5-7. For successful induction of immune tolerance, mucosal tissues play a significant role8-10. The epithelial layers that cover the Gut Associated Lymphoid Tissue (GALT) and Nasopharyngeal Associated Lymphoid Tissue (NALT) areas contain a subpopulation of specialized cells (microfold or M cells) that sample environmental antigens and present them to the adjacent immune cells11, 12. A number of studies now confirm that these cells play a crucial role in the generation of tolerance to a given antigen13-16. Reoviruses are segmented, double-stranded RNA viruses that bind and infect humans via mucosal surfaces using the viral coat protein, p?117, 18. We have demonstrated that fusion proteins consisting of p?1 fused to an antigen of choice can bind to M cells and generate a tolerogenic immune response to that antigen13-16, 19 . The ability of p?1-antigen targeting to induce tolerance has been demonstrated in both allergy and autoimmune models, including the mouse experimental autoimmune encephalomyelitis (EAE) model of MS 19,20 and oral allergy 14. The tolerance response is antigen specific, and due to the induction of anti-inflammatory cytokines and an increase in suppressive regulatory T cells (Tregs)13, 14, 19, 20. While lactic acid bacteria (LABs) are traditionally used for food production and their probiotic properties21, genetically engineered LABs have been produced by us and others to deliver immune-modulators to the host mucosa4, 22-25. To reduce the complexity and cost associated with producing therapeutic recombinant p?1- based proteins carrying multiple antigens, and to improve the mucosal pharmacokinetic and pharmacodynamic (PK/PD) properties following oral administration, our strategy is to deliver p?1-based proteins in the context of L. lactis. We believe L. lactis represents an ideal delivery system as it has ?Generally Regarded as Safe? (GRAS) status by the FDA, and is widely used in food applications26. This proposal is designed to validate a novel L. lactis platform (LacP?1) for rapid development and delivery of p?1-targeted immune-modulators. At the same time, we will also validate the first therapeutic candidate using this platform for the treatment of MS. The specific aims are to: 1) develop an L. lactis strain expressing a p?1 fusion protein containing three dominant MS auto-antigens (VTC-435); 2) demonstrate that oral administration of VTC-435 prevents neurological symptoms in the mouse EAE model, and 3) confirm that VTC-435 induces Tregs while reducing antibody levels in the EAE model.